This invention relates to an improved method for determining aldolase, EC 4.1.2.13, in serum and other biological fluids, and to related improved methods for determining other species.
Aldolase belongs to a group of enzymes, termed lyases, which reversibly cleave substrates into two compounds without hydrolysis. Aldolase splits the hexose fructose-1, 6-disphosphate (FDP) into the triose phosphates glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The term "triose" as used throughout this specification and claims is limited to the two species glyceraldehyde (GA) and dihydroxyacetone (DHA). The enzyme is present in virtually all cells of the body, especially in the muscles. Circulating blood normally contains comparatively little aldolase. Serum levels of aldolase increase in conditions which involve cell destruction, and it is believed that the normal level arises from physiologic breakdown of tissue. Clinically, the measurement of aldolase is useful, for example, in cases of muscular diseases such as progressive muscular dystrophy in which serum aldolase levels rise to five or ten times the upper limit of normal, and in prostate cancer in which the elevation of serum aldolase levels is less pronounced. Aldolase is also a commercial product, and tests are required for determining aldolase levels during the isolation of the enzyme.
Four well-known approaches to determining aldolase in serum (or equivalently, in plasma), by measuring the cleavage products of FDP, have been proposed. As set out in the article on aldolase by Bruns & Bergmeyer, in Methods of Enzymatic Analysis, ed. H. S. Bergmeyer (New York 1965), p. 724, the methods are as follows:
(1) DETERMINATION OF THE ALKALI LABILE TRIOSE PHOSPHATE FORMED. Meyerhoff & Lohmann, Biochem. Z. 271, 89 (1934) and 273, 73, 413 (1934).
(2) COLORIMETRIC ESTIMATION OF THE TRIOSE PHOSPHATE FORMED BY THE METHOD ORIGINALLY DESCRIBED FOR LACTATE. Dounce and Beyer, J. Biol. Chem, 173, 159 (1948).
(3) MEASUREMENT OF ACTIVITY BY THE SPECTROPHOTOMETRIC METHOD OF Warburg. Warburg and Christian, Biochem. Z. 314, 399 (1943).
(4) another colorimetric method in which the dinitrophenylhydrazones of the free trioses are determined. Sibley and Lehninger, J. Biol. Chemistry 177, 859 (1949).
A fifth approach, by Jagannathan et al, Biochem. J. 63, 94 (1956), adds hydrazine to the incubation mixture and measures absorbance at 240 nm.
Meyerhoff and Lohmann's early studies were made largely on aldolase obtained from muscle extract dialysates. The high blanks, variability and technical difficulties of alkali labile phosphorus determination have led not only to its being ignored as a routine method for determining aldolase in serum or other biological fluids, but also to numerous proposals for replacing it as a primary standard. No completely satisfactory primary or secondary standard has yet been devised. See Beck, J. Biol. Chem, 212, 847 (1955). Among other things, Meyerhoff and Lohmann found that the enzyme is active between pH's of about 6 and 10 (in a carbonate-bicarbonate buffer), and that it is not influenced by certain materials, such as iodoacetic acid, sodium fluoride and sodium oxalate, which are known to inhibit some other enzymes. In later work (Bull. Soc, Chimie Biol. 20, 1033) (1938)), Meyerhoff used hydrazine to fix the relative proportions of the triose phosphates GAP and DHAP in a 1:1 ratio to permit their polarimetric determination. Fixing the proportions of GAP and DHAP is necessary because certain enzymes which may be present in biological fluids selectively catalyze destruction of one of the trioses. In particular, triosephosphate isomerase (TPI), EC 5.3.1.1, which has a wide distribution in animal tissues, catalyzes the conversion of GAP to DHAP.
The colorimetric method of Dounce and Beyer degraded DHA and GA to acetaldehyde and added p-hydroxydiphenyl to produce a color, but their procedure suffered from frequent and variable high blanks and from the inconvenience of using concentrated sulfuric acid and heat. After the publication of the Sibley and Lehninger method, these workers modified their method by reducing the concentration of FDP substrate, adding hydrazine and adding collidine buffer pH 7.2. Dounce et al, J. Biol. Chem. 185, 769 (1950).
The method of Warburg and Christian, and other similar methods using coupled enzyme systems, suffer from several disadvantages, not the least of which is their need for an expensive ultraviolet spectrophotometer.
The colorimetric method of Sibley and Lehninger, with or without minor variations, is widely used for the determination of aldolase in serum. Sibley and Lehninger carried out the incubation of sample and FDP in Tris buffer at pH 8.6 and added hydrazine to the enzymatic incubation mixture to prevent the enzymatic conversion of GAP and DHAP through the action of TPI. After an alkaline hydrolysis step, they added an acidified 2,4-dinitrophenylhydrazine reagent to the alkaline hydrolysis mixture, then added more alkali to develop a characteristic color. The colored product is presumed to be hydrazone, and will be so characterized herein.
Some later investigators, such as Dounce et al, J. Biol. Chem., 185, 769 (1950), modified the sibley and Lehninger procedure by using a collidine buffer at a pH of 7.2 for the incubation mixture, and adding iodoacetate to the enzymatic incubation mixture to reduce side reactions and to lower the absorbance readings of the blanks used in the procedure. Friedman and Lapan, J. Clin. Lab. Med. 51, 745 (1958), recommended the use of DHA as a (secondary) standard, and the reporting of aldolase values in "DHA units".
Although the methods based on Sibley and Lehninger's method are regarded as the most reliable and suitable for routine laboratory use, all of these methods suffer from a lack of sensitivity, reliability and accuracy, from high blanks, from the instability of the colored compounds measured, and from the difficulty of preparing reliable standard solutions.